Abstract Rice ( Oryza sativa L.) agriculture of the southern United States is plagued by strong biotic competition with several species in the Echinochloa genus. Despite clear genomic differences between barnyardgrass ( Echinochloa crus-galli (L.) P. Beauv.] and junglerice ( Echinochloa colona (L.) Link], the two major Echinochloa agricultural weeds are nearly indistinguishable phenotypically. This inability to reliably differentiate the species has led farmers to treat the group as a single species, often resulting in ineffective weed control efforts. In this study, we first develop a simple chloroplast-anchored PCR-based restriction enzyme assay to differentiate between E. colona and the other Echinochloa species of agricultural concern. Applying this assay, we identify a strong bias towards E. colona in 2024 rice field collections from eastern Arkansas. Finally, we evaluate anecdotal reports of interspecific hybridization between species and find no evidence. Despite the drawbacks of the maternally inherited nature of the chloroplast, the availability of this species determinant assay will help USDA and academia extension agents and stakeholders to make educated, species-specific decisions about precision chemical weed control and field management.

4-Hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27, HPPD), as a key enzyme for tyrosine decomposition, is a highly regarded herbicide target. In this work, a series of cyclohexenone derivatives containing a substituted pyrazole group were designed and synthesized by active fragment splicing method for screening new HPPD inhibitors. The bioassay results indicated that some target compounds exhibited noticeable inhibitory activity against Capsella bursa-pastoris (CB), Portulaca oleracea (PO), Echinochloa crus-galli (EC) and Setaria viridis (SV). As an outstanding representative, compound 8n showed excellent inhibitory activity against four tested weeds with the rates more than 90% at 225 active ingredient per hectare (a.i./ha) dosage, especially with a rate more than 90% against CB at 75 a.i./ha dosage, comparable to the control herbicide mesotrione. Compound 8n also demonstrated satisfactory safety for the crops wheat, maize, sorghum and rice. Furthermore, the molecular docking, molecular dynamics (MD) simulations and frontier molecular orbital analysis revealed that compound 8n had strong interaction with the target protein AtHPPD, which could tightly bind with amino acid residues in the active pocket to generate its herbicidal activity. This study suggested that compound 8n could be used as a valuable lead molecular structure for the design of novel HPPD inhibitors, while providing an important foundation for the development of new herbicides. © 2025 Society of Chemical Industry.

The fall armyworm Spodoptera frugiperda (J. E. Smith, 1797) has evolved resistance to different Bt maize technologies carrying Bacillus thuringiensis genes. One hypothesis for the rapid evolution of resistance may be associated with the species' ability to multiply and survive on alternative host plants, especially during the off-season of the main crops. The aim of this study was to evaluate the biological parameters and fertility life table of susceptible (SS), heterozygous (RS) and resistant (RR) strains of S. frugiperda to Bt YieldGard VT PRO maize when kept in summer-winter cover crops. Based on the results, S. frugiperda did not survive on weeds Barnyardgrass (Echinochloa crus-galli). SS, RR, and RS larvae fed on white oats (Avena sativa), ryegrass (Lolium multiflorum), maize (non-Bt isoline) showed an egg to adult generation of approximately 30 d. The RS strain performed better than both the SS and RR strains, this is consistent with the individuals' high heterosis index. In addition, the insects reared on these hosts showed the highest reproductive rates (Ro = 496.2 to 578.1) and growth rates (rm = 0.143 to 0.185). In contrast, sorghum (Sorghum bicolor) or Persian clover (Trifolium resupinatum) prolonged the cycle by 4 d and reduced egg-to-adult viability parameters by 13%, larval weight (0.057 to 0.74 mg), pupal weight (0.36 to 0.86 mg), Ro (196.4 to 324.0), and rm (0.104 to 0.122). However, it is worth noting that all the host plants evaluated, except for Barnyardgrass, can serve to multiply S. frugiperda in the field and favor the reestablishment of the insect during the corn off-season and, consequently, accelerate the evolution of the pest's resistance.

Actinomycetes and fungi have been found to be important sources of bioactive natural products. Evaluation the diversity of culturable insect-asscociated microorganisms, and screening strains with bioactive activity holds a promising for the development of lead compounds. Here, the diversity, antimicrobial and herbicidal activities of culturable actinomycetes and fungi from Hermetia illucens was investigated for the first time. A total of 34 strains of actinomycetes and 27 strains of fungi were isolated from the gut and body surface of H. illucens. Among them, Streptomyces (97.1%) and Geotrichum (14.8%) were the most abundant genera in actinomycetes and fungi, respectively. The herbicidal bioassay showed that the fermentation broth of 3 actinomycetes and 13 fungi presented good inhibition activity (> 50%) against Echinochloa crusgalli and Abutilon theophrasti. The antibacterial bioassay showed that the crude extracts from 10 strains of actinomycetes (29.4%) displayed antibacterial activity against at least one pathogenic bacterium with the inhibition zone diameter (IZD) of 7.0-22.0mm at the concentration of 30µg/6mm filter paper disc. Especially, the strain of S. tritolerans MFT2 exhibited strong antibacterial activity against S. aureus with the IZD of 22.0mm, which were comparable to that of the positive gentamicin sulfate (IZD = 24.7mm). Total of four known compounds were isolated from MFT2 by column chromatography, and were further identified as gilvocarcin M (1), gilvocarcin V (2), γ-valerolactone (3), and chrysomycin C (4) based on NMR and MS. Among them, compounds 1-2 exhibited moderate antibacterial activities against P. syringae pv. actinidiae with the IZD of 10.3 and 13.2mm respectively at the concentration of 30µg per disc, which were weaker than that of gentamycin sulfate (IZD = 25.7mm). Furthermore, both compounds 1 and 2 exhibited moderate phytotoxicity against E. crusgalli with inhibition rate of 56.7% and 44.1% at a concentration of 100µg/mL, respectively. In conclusion, the rich sources of actinomyces and fungi associated with H. illucens offers promising avenues for discovering novel antimicrobial agents and bioherbicides.

Background: Minor millets, with their small size and hard husk, pose challenges in dehulling that affect overall yield and utility. Pretreatment methods were employed to facilitate more efficient dehulling. Methods: The current study was undertaken to assess the dehulling efficiency and evaluate the impact of various pre-treatments on the spatial and functional properties of barnyard millet (Echinochloa crusgalli L.). The grains were subjected to abrasive dehulling [A] for 2[A2], 4[A4], or 6[A6] minutes followed by centrifugal dehulling and the efficiency indices were calculated. Result: A2 and A4 samples exhibited superior milling efficiency and whole kernel recovery reflecting better grain integrity after processing. The A6 treatment resulted in highest head rice yield with intact grains whereas the control has lower retention and reduced dehulling performance. Spatial analysis of the grain revealed that A4 has the largest grain dimensions compared to other samples. Functional property evaluation indicated that A6 had the highest water holding capacity and swelling power making it ideal in developing rehydrated formulations, A4 was more appropriate for processed foods applications, A2 has good hydration properties, while the control sample was best suited for snack preparation. Among the pre-treated samples A2 sample was reported significantly dominant among the other pre-treated samples.

The reliance on chemical weed control in No-Tillage (NT) rice systems often leads to ecological changes in weed community composition and raises concerns regarding crop safety. This research aimed to evaluate the dynamics of weed community shift (SDR) and assess crop tolerance following the sequential application of glyphosate (pre-plant) and a mixture of cyhalofop-butyl + ethoxysulfuron (post-emergence). The experiment was conducted from May to October 2025 at the Experimental Farm of Universitas Padjadjaran, using a Split-Plot Design with three replications. The results indicated a significant weed composition shift from a community dominated by broadleaves (Limnocharis flava and Salvinia molesta) before application to a grass-dominated community (Echinochloa crus-galli and Leptochloa chinensis) at 3 weeks after application (WAA). Regarding crop tolerance, transient phytotoxicity symptoms in the form of leaf chlorosis were observed at 1 WAA, particularly in the highest dose treatment (1.75 L/ha). However, the rice plants demonstrated a rapid physiological recovery mechanism, with symptoms disappearing completely by 3 WAA. This recovery was confirmed by the vegetative growth response, where the number of vegetative tillers showed no significant inhibition compared to the manual weeding control during the active tillering phase. These findings suggest that sequential application of these herbicides effectively manages the weed spectrum shift and remains agronomically safe due to the crop's high resilience.

The sunflower is part of the order Compositales (Asterales), the family Compositae (Asteraceae), is native to Central and North America and spread throughout the globe, especially for its oil. It is a plant that is cultivated on large areas in our country, especially in Dobrogea, the Romanian Plain and the West. The development and implementation of a complex of measures and methods for combating weeds in agricultural crops requires a thorough study of their lifestyle and behavior in different climatic conditions, due to the fact that weeds have different biological properties compared to the crop plant. In the pedoclimatic conditions specific to the area at NARDI - Fundulea, the sunflower crop presents a high weeds infestation (75%), a weeds spectrum and a dominance specific to the area. In the experimental field, the sunflower crop had characteristic weed species, the most representative being: monocotyledonous- Setaria viridis, Echinochloa crus-galli, Sorghum halepense and annual dicotyledons – Amaranthus retroflexus,Chenopodium album and Polygonum convolvulus.The study and control of weeds is an important research direction, because they cause damage that can affect production by up to 35% - 70%. Effective weed management is one of the many critical components of sunflower production. The purpose of the research was to identify weeds, crop selectivity and combat the weed species present by applying herbicide treatments, with the objective of broadening the spectrum of control, synergism, persistence and without negative impact on the environment. The herbicide treatments must be correlated with the infestation degree of weeds, the spectrum and dominance of weeds, the time of application, the technical potential for efficacy, the local climatic conditions from Fundulea.of weeds, the time of application, the technical potential for efficacy, the local climatic conditions.

Natural rosin derivatives offer a promising scaffold for developing low-toxicity herbicides to address environmental concerns triggered by synthetic agrochemicals. In this research, a series of dehydroabietyl-hydrazide-based derivatives (3a-l) were synthesized using dehydroabietic acid as a lead compound. The herbicidal activities of these derivatives were evaluated against Echinochloa crusgalli, Sorghum halepense, Portulaca oleracea, and Amaranthus caudatus L. weeds. The result indicated that 3e exhibited broad-spectrum activity, particularly against barnyard grass root growth, with 91.0% inhibition at 100 μg/mL, outperforming the commercial herbicides naptalam (NPA) and glyphosate. Subsequently, the physiological indicators were employed to reveal the mode of action of 3e, and the result suggested that 3e significantly inhibited peroxidase enzyme activity, reduced the level of growth-promoting hormones, and increased the inhibitory hormone content, ultimately disrupting endogenous hormone homeostasis. Molecular docking demonstrated that 3e stably bound to the POD (-7.73 kcal/mol) via synergistic hydrogen bonding and hydrophobic actions. Crop safety assessments showed that 3e presented lower toxicity to wheat, corn, rice, and cotton seedlings at a high concentration of 200 g a.i./ha. The biosafety assessment evaluation also suggested that 3e showed lower toxicity to zebrafish, with 96 h median lethal concentration values of 33.9 μg/mL. Overall, these findings suggested that 3e could serve as a natural herbicide agent for controlling E. crusgalli, offering a sustainable weed management strategy.

The research was conducted in the Primorsky Territory at the experimental base of the Far Eastern Research Institute of Plant Protection in 2022–2024. The sensitivity of 15 weed species to the Kelvin Plus herbicide was studied in a vegetation house. Visual observations of their development after the treatments were carried out. Symptoms of herbicide action and the time of their manifestation were noted. It was found that the following are highly sensitive to the herbicide: Setaria glauca , Echinochloa crusgalli , Eriochloa vilosa , Amar а nthus retroflе xus , Elsholtzia cristata , Commelina communis , Hibiscus trionum , Siegesbeckia pubescens, and Sonchus arvensis . Sensitive plants include Polygonum persicaria , Chenopodium album , Acalypha australis , Cirsium setosum and Ambrosia artemisiifolia . Abutilon theophrasti was resistant to the drug's action. An assessment was made of the biological and economic efficiency of the herbicide Kelvin Plus at a consumption rate of 0.4 kg/ha when applied in the 5–6 and 7–8 leaf phases of corn. From the moment of application until harvest, regardless of the timing of treatment, the herbicide almost completely destroyed perennial dicotyledons, annual cereals and dicotyledons, including Ambrosia artemisiifolia , which dominated the crops in terms of growing aboveground mass. The high efficiency of applying the Kelvin Plus herbicide in the 5–6th leaf phase of the crop allowed increasing the yield by an average of 43.9 c/ha, with the yield in the control being 9.0 c/ha. A significantly smaller yield was preserved with late herbicide application – 24.5 c/ha. The interval between treatments was up to 20 days. During this period, weeds seriously competed with the crop, which impacted the resulting grain yield.

Elegance in simplicity: Hollow mesoporous silica inhibits pyraclonil metabolism to boost its herbicidal efficacy on Echinochloa crus-galli

Being a plant cultivated maize frequently becomes weedy at levels considered dangerously high. The plant, having requirements especially for water, benefits to the greatest extent from the segetal species, namely from the "summer" group. The determinations regarding weed species targeted areas cultivated with maize on private farms. The inventories carried out under these conditions are becoming important today, both for the perspective of weed conservation from an ecological perspective, and as their effective response to the control methods that are taken. The determinations resulted in a total of 22 species. Of these, 13 were part of the annual dicotyledons, 6 were perennial dicotyledons and 2 species were annual monocotyledons. The entire plant spectrum was part of 13 botanical families, the best represented being Poaceae. The species were then grouped according to density and constancy, and under these conditions the dominant species was Echinochloa crus-galli L./P. Beauv. With the help of this data, appropriate decisions can be made to control the entire weed spectrum in maize, especially for the dominant ones. The inventory of weeds in maize thus also acquires a clearly practical character, with the help of which some levels of economic control-fighting intervention are also established.

The persistent challenge of weed competition in agriculture necessitates the development of novel herbicides with enhanced efficacy and improved physicochemical properties. This study employed a molecular hybridization strategy to optimize tiafenacil by incorporating cyclopropyl and cyclobutyl moieties into pyrimidinedione scaffolds, aiming to discover more effective protoporphyrinogen oxidase (PPO)-inhibiting herbicides. Thirty-four novel pyrimidinedione derivatives were synthesized and evaluated for post-emergence herbicidal activity. Structure-activity relationship (SAR) analysis revealed that cyclopropyl derivatives consistently outperformed cyclobutyl analogs, with methyl esters providing optimal activity. In the greenhouse assay, compound FM-1688 (6aa) demonstrated exceptional in vivo herbicidal efficacy, achieving 100% control of broadleaf weeds (Zinnia elegans and Abutilon theophrasti) at 2.34375 g active ingredient (a.i.)/ha and 80% control of grass weeds (Echinochloa crus-galli) at 9.375 g a.i./ha, significantly surpassing the commercial standard tiafenacil (which achieved only 40% control of E. crus-galli at the same rate). Crucially, enzymatic inhibition experiments revealed that FM-1688 (half-maximal inhibitory concentration (IC50) = 5.95 nm) possesses only minimally greater in vitro target-site potency than tiafenacil (IC50 = 6.85 nm). This major discrepancy between in vivo efficacy and in vitro potency suggests the 'cyclopropyl-enhanced activity' is not a target-site (pharmacodynamic) effect. Molecular modeling supported this; docking studies revealed nearly identical binding poses and comparable binding energies (-8.6 kcal/mol for FM-1688 versus -8.7 kcal/mol for tiafenacil). Instead, density functional theory (DFT) calculations identified the true driver: FM-1688 possesses a significantly reduced molecular dipole moment (3.19 D) compared to tiafenacil (5.89 D), suggesting its superior greenhouse efficacy is driven by enhanced bioavailability and membrane permeability. The cyclopropyl-containing pyrimidinedione derivative FM-1688 represents a highly promising herbicide candidate. Its superior in vivo activity appears to be driven by cyclopropyl-enhanced pharmacokinetic properties (bioavailability) rather than enhanced target-site binding. This finding provides a new rationale for agrochemical design, warranting further development for sustainable weed management. © 2025 Society of Chemical Industry.

Abstract Which factors are the most important determinants of weed species composition in organic rice fields? Which species can be associated with the extreme values of the most important background variables? To answer these questions, we surveyed the weed flora along with 15 management and 3 environmental variables in 42 organically managed rice fields in eastern Hungary. Filamentous algae, Chara vulgaris , Chara braunii , Schoenoplectus mucronatus , Elatine triandra , and Echinochloa crus-galli were the most dominant weeds. Using a minimal adequate model containing 4 terms with significant net effects, 48.3% of the total variation in weed species data could be explained. Farm holding size (correlated with sowing type, shift crop, organic manure and water depth) was found to be the most important explanatory variable, which was followed by hand weeding, tillage type (correlated with tillage depth, shift crop and supplementary nutrients) and rice cover. Farm holding size was negatively associated with most of the aquatic plants suggesting its adverse impact on farmland biodiversity. Hand weeding appeared to be efficient against Rumex stenophyllus and Echinochloa crus-galli , but it was tolerated by Cyperus difformis . Perennial weeds like Persicaria amphibia and Bolboschoenus planiculmis were more abundant in sites without soil inversion and Lemna aequinoctialis seemed to be most tolerant for the shading effect of crop canopy. The responses of weed species to the studied variables provide new information about the assembly rules of aquatic plant communities, and our findings also can be used to optimise non-chemical weed control strategies in organic rice production.

Photosystem I (PS I) is a plastocyanin-ferredoxin oxidoreductase within the photosynthetic electron transport chain and represents a key target for herbicide development. Pyridinium salt compounds A and B were designed and synthesized with active substructural insertion and combination strategies. Bioassays revealed that most compounds exhibited > 70% inhibition of growth in Portulaca oleracea and Echinochloa crusgalli when applied to germinated seeds at a concentration of 10 μg mL-1. Compound B8 demonstrated 90-100% postemergence herbicidal activity on E. crusgalli, Eleusine indica, Setaria viridis, Amaranthus retroflexus, P. oleracea and Abutilon theophrasti. Meanwhile, B8 not only demonstrated electrochemical characteristics associated with PS I inhibitory activity and a negatively charged surface, but also promoted the accumulation of reactive oxygen species in Portulaca oleracea roots. At a concentration of 0.25 mM, B8 exhibited superior safety profiles in zebrafish embryos compared to paraquat. Furthermore, density functional theory calculations corroborated the redox reactivity and molecular orbital properties of B8, showed strong consistency with experimental observations. Compound B8 had herbicidal activity similar to paraquat with lower toxicity to zebrafish embryos. Therefore, B8 represents a promising lead candidate for new PS I targeted herbicides. © 2025 Society of Chemical Industry.

Peatlands, as globally important carbon sinks, are highly sensitive to human disturbances. The direct discharge or use of domestic sewage with different treatment gradients as peatland replenishment water affects plant communities, but how the plant community structure and biomass change and what drives these changes remain unclear. This study simulated domestic sewage input to a sedge-dominated peatland in the Changbai Mountain region of Northeast China, explored changes in plant community structure and aboveground biomass and revealed the key factors influencing these plant characteristics. The results revealed the following: (1) Reclaimed water treatment (Z) had little effect on the plant community structure; treatments with 50% tap water and 50% domestic sewage (H) and domestic sewage (W) caused rapid expansion of Poaceae plants, with Echinochloa crus-galli (L.) P. Beauv. occupying dominance. (2) Domestic sewage input increases plant diversity, which is driven jointly by soil nutrients and aboveground plant nutrients. (3) The H and W treatments significantly altered aboveground biomass, which was positively correlated with soil nutrients, aboveground plant nutrients, and diversity, whereas the Z treatment had little effect. This research provides scientific support for the control of domestic sewage discharge and whether reclaimed water can be used for ecological water replenishment in peatlands. It holds significant practical value for the scientific management of peatlands, the maintenance of carbon sink functions, and the mitigation of climate change.

Protoporphyrinogen oxidase (PPO) inhibitors represent an important research focus in herbicide development due to their high efficacy. Building on our previous identification of 3-(pyridin-2-yl)benzothiazol-2-one derivatives as novel PPO inhibitors, which demonstrated potent activity against broadleaf weeds but showed limited efficacy against grasses, we employed a hybridization-substructure splicing strategy. This approach integrated the oxazinone moiety of flumioxazin to design novel 3-(pyridin-2-yl)benzothiazol-2-one derivatives targeting broad-spectrum herbicidal activity. Starting from 4-chloro-3-nitroanisole or 2-amino-6-methoxybenzothiazole, we prepared 26 novel 3-(pyridin-2-yl)-thiazolo[5',4':4,5]benzo[1,2-b][1,4]oxazine-diones and confirmed their structures by proton (1H)-nuclear magnetic resonance (NMR), carbon-13 (13C)-NMR, and high-resolution mass spectrometry (HRMS) analysis. Petri dish assays revealed that compounds 9b, 9d and 9e exhibited potent herbicidal activity against both Portulaca oleracea and Echinochloa crus-galli at 10 μg mL-1. Post-emergence evaluation (75 g active ingredient (a.i.) ha-1) showed that compound 9b achieved 100% control efficiency against Amaranthus retroflexus, Datura stramonium, Abutilon theophrasti, Solanum nigrum and Setaria viridis, while demonstrating superior crop safety in Zea mays and Oryza sativa. Compound 9b treatment induced a dose-dependent increase in protoporphyrin IX accumulation in plants, reaching levels of 1.37 ± 0.35 μg g-1 and 3.23 ± 0.27 μg g-1 at concentrations of 12.5 μg mL-1 and 25 μg mL-1, respectively. A new class of oxazinone-containing 3-(pyridin-2-yl)benzothiazol-2-one PPO inhibitors was developed, demonstrating promise as broad-spectrum herbicide candidates. These findings provide valuable insights for future PPO herbicide research and development. © 2025 Society of Chemical Industry.

Multi-gene metabolic detoxification conferring cyhalofop-butyl resistance in Echinochloa crus-galli (Barnyard grass).

Variability and spatial distribution of ALS-inhibitor resistance mechanisms in Brazilian Echinochloa crus-galli.

The organization of leaf mesophyll at 8 species of panicoid grasses with NADP-ME type C4 photosynthesis was studied using a light microscope on transverse and longitudinal sections. Plants of natural communities (Arundinella hirta, Spodiopogon sibiricus), weeds (Echinochloa crus-galli, Setaria viridis) and cultivated grasses (Echinochloa colonum subsp. edulis, Pennisetum americanum, Sorghum × drummondii, Zea mays) were distinguished. Anatomical studies were carried out in the middle part of the leaf blades and the upper third of the leaf sheaths located in the middle of the generative shoots. Among the mesophyll cells of the leaves, the cells of the crown sheath radially adjacent to the Kranz cells and the cells of the intercrown zone located between them were distinguished. It is shown that the mesophyll of the leaves of the considered grasses consists of cells of simple and complex cellular shapes. Cellular cells meet more often in the crown sheath compared to the intercrown zone. In leaf blades of grasses of natural cenoses, especially at Arundinella hirta, they are the most numerous, with a large number of well-defined sections. In weeds and cultivated grasses, the participation of cellular cells decreases; in their majority they consist of 2–4 sections and are often characterized as weakly cellular. In leaf sheaths the share of assimilative cells of simple form increases, most of all in weeds and cultivated grasses.

Echinochloa crus-galli and E. crus-galli var. mitis are two of the most troublesome rice weeds. Florpyrauxifen-benzyl is one of the most important post-emergence rice herbicides that has been pervasively applied in many countries since 2018. We collected 70 E. crus-galli and 158 E. crus-galli var. mitis populations from rice fields in eastern China in 2022 and tested their sensitivities to florpyrauxifen-benzyl through whole-plant bioassays. A total of 21 days after treatment with florpyrauxifen-benzyl label dose (36 g ai ha−1), 71.4% of E. crus-galli and 70.9% of E. crus-galli var. mitis populations were completely controlled. The GR50 doses (doses causing 50% fresh weight reductions in aboveground parts) of florpyrauxifen-benzyl applied to E. crus-galli populations ranged from 1.4 to 36.9 g ai ha−1, with a baseline sensitivity dose of 4.9 g ai ha−1; those for E. crus-galli var. mitis populations ranged from 1.3 to 97.6 g ai ha−1, with a baseline sensitivity dose of 5.0 g ai ha−1. No significant differences between E. crus-galli and E. crus-galli var. mitis were found in GR50 values. Among 70 E. crus-galli populations, 61.4%, 35.7%, and 2.9% showed no, low, and moderate resistance to florpyrauxifen-benzyl, while among 158 E. crus-galli var. mitis populations, 54.4%, 36.1%, 1.9%, and 1.9% showed no, low, moderate, and high resistance to florpyrauxifen-benzyl, respectively. Moreover, the frequency of florpyrauxifen-benzyl-resistant populations of E. crus-galli var. mitis tended to be higher in southwestern areas.